Cryptococcal immunity and immunostimulation.
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Animals↗
Biomedical subjects
J W Murphy
Publications and source records attributed to J W Murphy.
Immunomodulation in the mycoses.
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Animals↗
A method for hyperthermic treatment of mouse skin.
The Sencar mouse skin system is a recognized model for tumour initiation, promotion and progression. The current interest in the effect of hyperthermia on this multi-stage tumorigenesis model prompted the need for a technique to accurately heat a section of dorsal skin of a large number of mice for 30 min per heat treatment. In the technique described, experimental groups of 25 female Sencar mice were treated at 7-8 weeks of age under general methoxyflurane anaesthesia. Treatment consisted of the application of initiating and/or promoting agents with or without hyperthermia. For hyperthermic skin treatments, each group of mice was placed onto a platform in a water bath so that the dorsal skin of the mice was in contact with 44 degrees C temperature controlled water.
Anesthesia, General↗
Effects of Cryptococcus neoformans-specific suppressor T cells on the amplified anticryptococcal delayed-type hypersensitivity response.
Cell-mediated immunity is an important host resistance mechanism against Cryptococcus neoformans, the etiological agent of cryptococcosis. Previous studies from our laboratory have shown that the anticryptococcal cell-mediated immune response as measured by delayed-type hypersensitivity (DTH) is down-regulated by a cascade of antigen-specific T suppressor (Ts) cells. Recently, we have identified a population of CD4 T cells that up-regulate the anticryptococcal DTH response (Tamp cells). The Tamp cells are found in the spleens of donor mice at 6 days after immunization with cryptococcal antigen, and they amplify the anticryptococcal DTH response when transferred to syngeneic recipients at the time of immunization of the recipients. In this study, we determined the effects of C. neoformans-specific Ts cells on the induction of the Tamp cells in the Tamp cell-donor mice and on the induction and expression of the amplified anticryptococcal DTH response in the Tamp cell-recipient mice. When cryptococcal-specific Ts1 cells were given at the time of immunization of the Tamp cell-donor mice, induction of Tamp cells was inhibited. In contrast, when Ts1 cells were given at the time of adoptive transfer of Tamp cells, the recipients displayed amplified DTH responses, indicating that Ts1 cells do not affect the Tamp cells' function once the Tamp cells have been produced. C. neoformans-specific Ts2 cells given at the time of either immunization or footpad challenge of the Tamp cell-recipient mice did not alter, to any measurable extent, the amplified DTH response. These results indicate that in addition to amplifying the anticryptococcal DTH response, Tamp cells may protect the anticryptococcal TDH cells from suppression by C. neoformans-specific Ts cells, much like contrasuppressor cells do in other systems. However, further characterization of the Tamp cells revealed that they are not adherent to Viscia villosa lectin, indicating that the anticryptococcal Tamp cells do not have this characteristic in common with contrasuppressor cells of other antigen systems.
Animals↗
Binding interactions of murine natural killer cells with the fungal target Cryptococcus neoformans.
Murine natural killer (NK) cells have been shown to inhibit the growth of the yeastlike organism Cryptococcus neoformans both in vivo and in vitro. An essential first step in NK cell-mediated damage of cryptococcal cells is the binding of the NK cell to the cryptococcal cell. The studies presented here focused on the binding event. Electron photomicrographs and three-dimensional reconstructions of NK cell-C. neoformans conjugates show that NK cells bind to cryptococci through many microvilli. This is in contrast to the broad membrane-membrane interactions which form the binding site of NK cell-YAC-1 tumor cell conjugates. NK cell binding to cryptococci is much slower than NK cell binding to YAC-1 targets. Maximal conjugate formation with cryptococcal targets is reached after 2 h, whereas maximal conjugate formation with YAC-1 targets is obtained after 20 min. Once maximum NK cell-C, neoformans conjugate formation is obtained, another 4 h is required before damage to the cryptococcal cells can be detected with the CFU assay. These data indicate that the binding and action of NK cells on C. neoformans cells requires considerably more time than is necessary for similar events to occur in the NK cell-tumor cell model. NK cell membrane integrity is necessary for NK cells to bind to tumor targets, since some disruption of membrane integrity with 0.1 M dimethyl sulfoxide reduces conjugate formation and tumor cell lysis. In contrast, 0.1 M dimethyl sulfoxide did not diminish NK cell binding to cryptococcal targets; however, it significantly reduced cryptococcal growth inhibition. Although we have observed several differences in NK cell binding to the cryptococcal target compared with NK cell binding to tumor cell targets, there are some similarities in binding interactions of NK cells with the two different targets. Disulfide bonding appears to play a role in the binding of NK cells to both targets, since 5 mM 2-mercaptoethanol, a reagent that reduces disulfide bonds, prevented NK cells from binding to the tumor targets as well as the cryptococcal targets. Actin filaments, components of the cytoskeletal network, must be intact for NK cells to bind to YAC-1 cells or cryptococci. Taken together, our data confirm that binding of NK cells to the cryptococcal target is prerequisite to the stages that result in damage to the cryptococcal cell and that there are similarities and differences in NK cell-binding interactions with structurally different target cells.
Animals↗
Responses of murine natural killer cells to binding of the fungal target Cryptococcus neoformans.
Natural killer (NK) cells bind to and inhibit the growth of the fungal target Cryptococcus neoformans. Since C. neoformans is structurally and chemically distinct from the standard tumor cell target used in the model of NK cell-mediated cytotoxicity, this study was designed to investigate the NK cell response after binding to cryptococci. Transmission electron micrographs and three-dimensional reconstructions of NK cell-cryptococci conjugates demonstrated focusing of the NK cell centrioles and Golgi apparatus toward the cryptococcal attachment site. NK cell cytoskeletal changes after cryptococcal binding were confirmed by immunofluorescence studies in which NK cells were allowed to bind to cryptococci in Mg2(+)-containing, Ca2(+)-free medium. One hour after the addition of Ca2+ to the preformed conjugates, the bound NK cells demonstrated a significant increase in the percentage of microtubule organizing centers focused toward the cryptococcal binding site. Colchicine, a drug that inhibits microtubule assembly, did not affect NK cell-cryptococci binding but abrogated NK cell-mediated cryptococcal growth inhibition, indicating that microtubule assembly, an important prerequisite for the secretory process, is not required for NK cell-cryptococci binding but is essential for inhibition of cryptococcal growth. In addition, the Ca2+ channel-blocking reagents, lidocaine and verapamil, did not affect NK cell-cryptococci binding but blocked the NK cell-mediated anticryptococcal activity, suggesting that a Ca2+ flux is essential for inhibition of cryptococcal growth. Considered together, these data indicate that NK cells respond to binding of a target cell that has a capsule and cell wall, in addition to a cell membrane, in a manner similar to that seen following binding to target cells that are surrounded by only a cell membrane; however, the response of the NK cells to the binding of C. neoformans is slower and possibly less efficient than the response after tumor cell binding.
Animals↗
Murine natural killer cells are fungicidal to Cryptococcus neoformans.
Murine natural killer (NK) cells have been shown to bind to and inhibit the growth of Cryptococcus neoformans in vitro and to contribute to clearance of the organism in vivo. However, it is unclear whether NK cells actually kill cryptococci or simply inhibit proliferation of the fungal target. Therefore, the studies presented here were designed to determine whether NK cells are fungicidal to C. neoformans targets. C. neoformans viability was determined on the basis of the metabolic function of two different enzyme systems, as measured by the two vital stains MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and fluorescein diacetate. Cryptococcal viability, as determined by vital stains, was compared with cryptococcal proliferation, as measured by microcolony formation in agarose at the individual cell level and by CFU counts or extinction dilution analysis in the total cell suspension. Initial comparisons of the vital stains and proliferation assays indicated that these methods effectively distinguished between live and heat-killed cryptococci at the individual cell level and in the total cell suspensions. After cryptococci were incubated with murine NK cells for 18 h, vital stains demonstrated that at the single conjugate level and in the total cell suspension, NK cells kill bound C. neoformans target cells. In addition, the numbers of dead cryptococci in the NK cell-C. neoformans suspensions as determined by the vital stains were comparable to the numbers of cryptococci that were unable to proliferate. Kinetics of NK cell-mediated C. neoformans binding and killing at the single conjugate level and in the total cell suspension were assessed by MTT staining at 2-h intervals after mixing effector and target cells, and the data support the concept that NK cell-C. neoformans binding precedes cryptococcal death. Furthermore, unbound, dead fungal cells were observed in the NK cell-C. neoformans suspensions after 18 h, suggesting that NK cell-C. neoformans interactions may involve both effector cell recycling and killing of unbound cryptococci by soluble cytotoxic factors. In conclusion, the results of these studies firmly establish that NK cells kill C. neoformans.
Animals↗
Early events in initiation of alternative complement pathway activation by the capsule of Cryptococcus neoformans.
The capsule of Cryptococcus neoformans is a powerful activator of the alternative complement pathway. This study examined the manner in which the cryptococcal capsule influences initiation of and early events in complement activation by C. neoformans. These studies examined the effects of the classical and alternative pathways on the kinetics and early sites for deposition of C3 fragments on encapsulated cryptococci, nonencapsulated cryptococci, and zymosan. The results showed that nonencapsulated cryptococci and zymosan are qualitatively and quantitatively similar in the manner in which they initiate complement activation. Both utilize the classical and alternative pathways. Initiation via the classical pathway occurs suddenly and simultaneously at sites distributed over the entire cell surface. Initiation of the alternative pathway by zymosan and nonencapsulated cryptococci is characterized by a lag of 6 to 8 min before appreciable amounts of C3 accumulate on the cells. Alternative pathway initiation by zymosan and nonencapsulated cryptococci occurs at a limited number of focal initiation sites that expand with alternative pathway amplification to cover the cell surface. Presence of the cryptococcal capsule blocks classical pathway initiation, which would normally occur at the cryptococcal cell wall, and produces an initiation that is dependent solely on the alternative pathway. Initiation of the alternative pathway by the cryptococcal capsule is characterized by a lag in C3 accumulation and the appearance of a limited number of focal initiation sites which resemble those observed when the alternative pathway is activated by zymosan and nonencapsulated cryptococci.
Complement Activation↗
Mechanisms of natural resistance to human pathogenic fungi.
Natural host-resistance mechanisms are essential first-line defenses against most mycotic agents; however, these defenses are often not sufficient for complete protection. The host relies on the immune responses to provide the additional antifungal activity necessary for maximum protection. In systemic mycotic diseases, the immune system must be functional to prevent the host from succumbing to the disease. Natural resistance mechanisms act together in a coordinated manner early in the disease process to either kill or prevent the fungal agent from proliferating and gaining entrance to other tissues. The early defensive measures of the natural effector mechanisms are usually sufficiently effective to provide time for the generally more effective immune defenses to develop. Together, the natural defenses and the immune defenses regulate each other through cytokine networks, and generally these systems provide the host with adequate protection against threatening mycotic agents.
Disease Susceptibility↗
A residency-based information system.
The medical literature has grown to an unmanageable size, and we need to develop information systems that provide better accessibility. An internal medicine residency program is an ideal setting for developing a system in which articles are screened for usefulness in clinical decision making and organized to be rapidly accessible and relatively inexpensive to use. In our residency training program, we use clinical problem-solving, journal club article review, and teaching conference preparation to generate appropriate articles from PAPERCHASE (MEDLINE) database searches. With the chief resident supervising the process, articles are then selected and organized in a computerized article file. The software program, Reference Manager (Research Information Systems, Inc., Encinitas, California), stores article citations and supports searches using standard, medical subject heading vocabulary key words. In an 18-month period, residents have collected more than 1800 references appropriate to clinical decision making and have established an article file that is now used daily as a medical information source.
Abstracting and Indexing↗
Cytoplasmic components of natural killer cells limit the growth of Cryptococcus neoformans.
Murine natural killer (NK) cell-mediated inhibition of growth of a yeast-like target cell, Cryptococcus neoformans, was completely abrogated by blocking the effector cell secretory process with monensin. Therefore, further studies were performed to determine the ability of various cytoplasmic fractions of NK cells to mediate inhibition of cryptococcal growth. Percoll-fractionated homogenates of rat LGL tumor cells demonstrated that the granule-containing fractions plus three additional sets of less dense cytoplasmic fractions displayed anti-cryptococcal activity; whereas only the cytoplasmic granule-containing fractions had cytotoxic activity against YAC-1 tumor cell and sheep erythrocyte targets. Maximal cryptococcal growth inhibition induced by LGL granules occurred after a 1 h incubation, required the presence of Ca2+ (1.0 mM) or Mg2+ (0.5 mM or 5.0 mM), and was completely abrogated in the presence of rabbit anti-LGL granule IgG. Cytolysin, the granule component which mediates tumor cell and sheep erythrocyte lysis, effectively limited the growth of cryptococci. Since Percoll gradient fractionation of the LGL homogenates demonstrated three separate peaks of anti-cryptococcal activity other than the granule peak, it is possible that the cytolysin-containing granules are not the only subcellular component of NK cells playing a role in inhibition of C. neoformans growth.
Animals↗
Characterization of a cell population which amplifies the anticryptococcal delayed-type hypersensitivity response.
Cell-mediated immunity to Cryptococcus neoformans can be detected by delayed-type hypersensitivity (DTH) to a culture filtrate antigen of C. neoformans. Recently, we have identified a population of cells in spleens of mice immunized with cryptococcal antigen that, when transferred to recipient mice at the time of immunization, amplifies the anticryptococcal DTH response. If the cell donor mice are treated with cyclosporin A during induction of the anticryptococcal DTH response, the amplifier cells are not induced, whereas the cells which transfer DTH (TDH cells) are induced. The purpose of this study was to characterize the amplifier cells with respect to their surface and functional properties and, in so doing, determine whether or not the amplifier cells are analogous to long-lived memory cells. We demonstrated that the amplifier cells were nylon-wool-nonadherent, antigen-specific, CD4 (L3T4+ Lyt-2-) T lymphocytes which appear in the spleens of mice 5 days postimmunization with cryptococcal culture filtrate antigen in complete Freund adjuvant. The amplifier T (Tamp) cells are not considered to be memory cells because they are relatively short-lived, being present 14 but not 18 days after the stimulating immunization. Moreover, the amplified anticryptococcal DTH response does not fulfill the criteria of the typical secondary immune (anamnestic) response in that the amplified response does not appear early relative to the appearance of the primary anticryptococcal DTH response, and it does not persist longer than the primary DTH response. We speculate that Tamp cells are not long-lived memory cells but rather act in a T-helper cell capacity to amplify the anticryptococcal DTH response.
Animals↗
T cell response to soluble cryptococcal antigens after recovery from cryptococcal infection.
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Antigens, Fungal↗
Effects of cyclosporin A on the cells responsible for the anticryptococcal cell-mediated immune response and its regulation.
Cyclosporin A (CsA), a potent immunosuppressive drug, was used to explore further the induction, expression, and regulation of lymphoid cells involved in the delayed-type hypersensitivity (DTH) response to cryptococcal antigen(s). We found that the induction of the cells responsible for DTH (TDH cells) was not affected by CsA, but their expression was inhibited in CsA-treated mice. The inhibition of expression of the TDH cells could not be attributed to the Cryptococcus neoformans-specific suppressor T (Ts) cells, even though the Ts cells were induced in CsA-treated mice. Instead, the suppressed expression of the TDH cells in CsA-treated mice was a direct effect of CsA or its products. Our studies with CsA also resulted in the first identification of a population of cells that significantly amplify the anticryptococcal DTH response. The amplifier cells were induced in mice that were given a primary immunizing dose of cryptococcal antigen in complete Freund adjuvant, and they amplified the anticryptococcal DTH response in recipient mice when they were transferred at the time of immunization of the recipient. The amplifier cell population was distinct from the TDH cells in that CsA inhibited the production of the amplifying cells but did not affect the induction of TDH cells. Amplification of the DTH response was a cell-mediated event, since cells but not serum from immunized mice mediated the amplified response in recipient mice. Thus, CsA enabled us to characterize anticryptococcal TDH and Ts cells further and to add to the immune cell circuit of the cryptococcal system a distinct population of cells that amplifies the anticryptococcal DTH response.
Animals↗
Clearance of Cryptococcus neoformans from immunologically suppressed mice.
To assess the effects of cryptococcal antigen-induced immunosuppression on a Cryptococcus neoformans infection, CBA/J mice were injected intravenously with saline or suppressive doses of cryptococcal antigen (CneF) at weekly intervals and were then infected with viable C. neoformans cells. By the second week after infection, the cryptococcal antigen-injected mice had suppressed anticryptococcal delayed-type hypersensitivity (DTH) responses compared with the responses of the saline-treated, infected control mice. In addition, the immunosuppressed mice had higher numbers of cryptococcal CFU cultured from their lungs, livers, spleens, lymph nodes, and brains than did the control animals. A direct correlation of suppression of the anticryptococcal DTH response and reduced clearance of cryptococci from tissues was also observed after mice were given a single intravenous injection of CneF and infected. To determine whether or not the cryptococcal antigen was specifically reducing the clearance of C. neoformans or had a more generalized effect, mice were injected with saline or suppressive doses of CneF, infected with Listeria monocytogenes, and then followed daily for 7 days for the clearance of L. monocytogenes from spleens and on day 7 for DTH reactivity to Listeria antigen. There were no differences between the saline- and CneF-treated mice with respect to anti-Listeria DTH responses or clearance of L. monocytogenes from spleens, indicating that CneF was not altering natural resistance mechanisms responsible for early clearance of L. monocytogenes, nor was the CneF influencing the induction of the acquired immune response which was responsible for the late clearance of the bacteria. Together, these data indicate that the specific suppression of this cell-mediated immune response induced by cryptococcal antigen reduces the ability of the animals to eliminate the homologous organism (C. neoformans) but not a heterologous infectious agent, such as L. monocytogenes.
Animals↗
Murine natural killer cell interactions with a fungal target, Cryptococcus neoformans.
Earlier investigations have shown that murine natural killer (NK) cells bind to and inhibit the growth of the fungal pathogen Cryptococcus neoformans in vitro and in vivo. To define the stages of NK cell-mediated inhibition of C. neoformans growth and the requirements for the completion of these stages, the events which lead to cryptococcal growth inhibition were compared with those previously elucidated for NK cell-mediated tumor cell lysis. Our data indicate that NK cell-cryptococci binding is a distinct event that precedes inhibition; is temperature independent, although it is slowed at 4 degrees C; and is Mg2+ dependent. In contrast to binding, NK cell-mediated cryptococcal growth inhibition is temperature, Mg2+, and Ca2+ dependent. The removal of Ca2+ by EDTA addition within 3 h after maximal NK cell-cryptococci binding significantly reduced cryptococcal growth inhibition, indicating that Ca2+ is required either late in the NK cell trigger stage or early in the inhibitory stage. These stages and requirements are similar to those previously demonstrated for the model of NK cell-mediated tumor cell lysis; however, the NK cell-cryptococci interactions are somewhat slower than the interactions which culminate in the lysis of the YAC-1 tumor cell targets. These results suggest that C. neoformans cells, although structurally distinct from the standard tumor cell targets, are capable of similar cell-to-cell interactions with NK effector cells as the tumor cell targets.
Animals↗
Immunoregulation in cryptococcosis.
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Animals↗
Therapeutic versus traditional foster care: theoretical and practical distinctions.
This article explores the conceptual and practical differences between traditional and therapeutic foster care. Most important is that therapeutic foster care is linked to an emerging philosophy referred to as postmodernism. If this philosophical basis is not understood, the thrust of therapeutic foster care may be seriously misconstrued, and a community-based style of treatment may be destroyed.
Community Mental Health Services↗